Taste sense is the sense generated by binding of a specific receptor present, particularly, on a surface of a tongue with a substance, when a substance is placed in a mouth. The taste sense of a mammal is constructed of five fundamental tastes, that is, a salty taste, a sour taste, a sweet taste, an umami taste and a bitter taste, and is considered to be formed by integration of these fundamental tastes. Currently, it is said that a salty taste and a sour taste are sensed via some ion channel-type receptors expressed on a cell membrane on a proximal side of taste cells present in taste buds on a surface of a tongue and, it is considered that an ion channel-type receptor consisting of PKD2L1+PKD1L3, which are a TRP channel family, functions, particularly, regarding a sour taste.
On the other hand, regarding a sweet taste, an umami taste and a bitter taste, it is considered that those tastes are sensed by signal transduction via a G protein coupled receptor (GPCR), which is a membrane protein present in taste cells, and a G protein coupling with it. Specifically, it has been revealed that a sweet taste is received with a heterodimer of T1R2+T1R3 (sweet taste receptor), an umami taste is received with a heterodimer of T1R1+T1R3 (umami taste receptor), and a bitter taste is received with about 30 kinds of molecules named as T2R family (bitter taste receptor).
The G protein is constructed of three subunits of α, β and γ. The state where α, β and γ subunits are connected is an inactive type and, when a taste substance binds to a G protein coupled receptor, GDP (guanosine 5′ diphosphate) which has been bound to an α subunit is substituted with GTP, resulting in an active type in which the G protein has been dissociated into a binding body of a GTP-α subunit and a β-γ subunit.
The construction of a transduction mechanism of taste sense information has not been completely clarified, but is generally understood as follows. That is, a popular opinion is that, first, when a taste substance is bound with a receptor of taste cells, a calcium concentration in cells is raised via an information transduction process through a second messenger (IP3, DAG) and the like in cells. A calcium ion supplied into cells then releases a neurotransmitter into a synapse to generate an action potential in nerve cells and, as a result, a taste sense signal starting from the receptor is transduced from a taste nerve to a brain, and the taste sense information is discriminated and determined. Recently, a theory is also being accepted, that a calcium ion opens a novel cation channel called TRPM5, and an inflow of a monovalent cation into cells causes depolarization.
Among the above-mentioned five fundamental tastes consisting of a salty taste, a sour taste, a sweet taste, an umami taste and a bitter taste, particularly, a sweet taste is a taste which is felt very tasty when a sugar content in blood is decreased. Since a sweet taste gives an image of an energy source including a sugar to a human and, further, causes a stronger feeling of satisfaction and a stronger feeling of happiness as compared with other fundamental tastes, and is deeply involved in emotion of a human, it is a taste central to determination of preference of beverages and foods.
However, a sweet taste has a higher minimum sensitivity (threshold) as compared with other fundamental tastes, and has a property that a sweet taste is sensed with difficulty in a small amount. On the other hand, when a sweet taste is too strong, this results in inducing an unhealthy image such as high calorie and obesity. For this reason, when beverages and foods with a sweet taste imparted thereto are produced, it is important to regulate an intensity of a sweet taste so that the sweet taste can be sensed by a human and preferably accepted.
An intensity of a sweet taste which is sensed when a human puts beverage and food, or the like into a mouth, has previously been verified mainly by sensory evaluation by a human. However, since sensory evaluation integrally assesses information sensed from the taste sense and the smell sense, it is difficult to verify to what extent a substance acts on the taste sense. Particularly, regarding a substance having a sweet taste not higher than a threshold, since a human cannot sense a sweet taste at a level not higher than a threshold, it is very difficult to verify an extent of a sweet taste. Further, it is very difficult to objectively assess a difference of a sweet taste sensitivity between substances. In the sensory evaluation or sensory test, a variation of evaluation between well trained assessors (called panel or panelist) cannot be neglected.
Then, it is also considered to isolate taste cells present in taste buds on a surface of a tongue, and using these isolated taste cells to assess a sweet taste intensity in place of sensory evaluation by a human. However, since taste cells isolated from those present in taste buds on a surface of a tongue are very weak in adhesion onto a culturing plate, and immediately die, they cannot be used in time-consuming assessment.
Since a sweet taste is a taste central to determination of preference of beverage and foods as described above, for example, if a substance acting on a sweet taste receptor to enhance a sweet taste can be identified, the advantageous effects such as improvement in a taste of beverage, foods and medicaments, reduction in a use amount of a sweetener, and reduction in ingested calorie can be attained. Therefore, great interest is given to such a sweet taste enhancing substance from the industrial world of beverages, foods, flavors and the like.
Then, as a new method of assessing a sweet taste intensity in place of sensory evaluation, there has been reported a method of objectively measuring and assessing a sweet taste intensity of a substance using a cell line actually expressing a sweet taste receptor.
Specifically, for example, in Example 11 of Patent Literature 1, there has been reported a cell strain generated by coexpression of hT1R2/hT1R3 by transfecting a linearized pEAK10-derived (Edge Biosystems) vector comprising an expression construct of hT1R2 (plasmid SAV2486), and a pCDNA3.1/ZEO-derived (Invitrogen) vector comprising an expression construct (plasmid SXV550) of hT1R3 into a Gα15-expressing cell strain (HEK-293 cell strain of Aurora Bioscience).
In Example 6 of Patent Literature 2, there has been disclosed a signaling system obtained by introducing a vector comprising a cDNA of hT1R2, a vector comprising a cDNA of hT1R3, a vector comprising a gene encoding a chimeric Gα protein, and a marker of a transfection efficiency pDsRed2-N1 (Takara Bio Inc.) into HEK-293T cells so that a DNA introduction ratio became a weight ratio (4:4:1:0.2) and an introduction amount became 4.6 to 5.5 μg, using a Lipofectamine 2000 reagent (Invitrogen), to coexpress hT1R2-hT1R3, and a chimeric Gα protein in HEK-293T cells.
In Example 7 of Patent Literature 3, there has been described a cell strain produced by transfecting a linear pIRES2-Puro vector (Clonetech) comprising a cDNA encoding hT1R3 into cells expressing Gα16gust44, then, transfecting a linear pcDNA4-TO vector (Invitrogen) comprising a cDNA encoding hT1R2, thereby, coexpressing hT1R2/hT1R3.